A variety of chemical-reaction methods for converting N-protected amino acids, including the C-terminal amino acid residues of peptides, to a corresponding thiohydantoin (TH) have been proposed TH formation is useful, for example, in C-terminal amino acid sequencing, where a C-terminal amino acid of a peptide is sequentially (a) converted to its corresponding TH, (b) cleaved from the remaining peptide, and (c) identified as to amino acid, e.g., by HPLC. Another use of such reaction methods is in preparing amino acid TH compounds for use as standards, e.g., in C-terminal sequencing.
The conversion of protected amino acids to corresponding amino acid TH's was first proposed in 1011 by T. B. Johnson et al. (J. Am. Chem. Soc., 48:103 (1911), and in 1926 by Schlack and Kumpf [Hoppe-Seyder's Z Phys Chem, 154:125 (1926)]. A number of improvements to this early method have since been proposed (see, e.g., Stark; Boyd et al., U.S. Pat. No. 5,051,368; Hawke, 1987; Hawke, U.S. Pat. No. 4,837,165; Hawke et al., U.S. Pat. No. 5,049,507; Inglis; Miller, 1988; and Miller, U.S. Pat. No. 4,935,494).
Stark's chemistry for making 2-thiohydantoins often results in low yields and derivatized products (i.e. acetylation and dehydration). Also, Hawke et al., (U.S. Pat. No. 5,049,507) propose a method of forming a peptidyl-TH in which an acyl-isothiocyanate moiety is used to generate the thiohydantoin. However, this method has the same limitations as are generally associated with acetylation of the terminal carboxyl group (Stark), e.g., degradation of the amino acid side chains of the peptide. Recent efforts to improve thiohydantoin-based methods have focused on improving the efficiency of forming the C-terminal activated ester. For example, Boyd et al. (U.S. Pat. No. 5,051,368) propose a method of forming a C-terminal thiohydantoin that employs N-substituted ketenimines, as generated by Woodward's Reagent K, to form the activated ester.